The nervous system consists of two major portions: the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS includes the brain and the spinal cord, and the PNS consists of all of the nerve tissue outside the CNS. The brain consists of the nervous tissue contained within the skull. The PNS is organized into nerves, bundles of nervous tissue that emanate from the CNS and extend throughout the body, where they provide pathways for signals travelling to and from the CNS portions (Encyclopaedia of human biology, volume 5 Ed Dulbecco).
The autonomic nervous system is that part of the PNS that regulates unconscious involuntary activities, such as the control of heart beat, movements of the digestive system, or glandular activities. It consists primarily of visceral efferent neurons that carry motor impulses to cardiac muscle, certain glands, and smooth muscles in blood vessels and organs of the thoracic and abdominal cavities.
The autonomic nervous system has two distinct anatomic and functional subdivisions: sympathetic and parasympathetic. The sympathetic neurons emerge from thoracic and lumbar regions of the spine, innervates smooth muscles of the arteries. Just as arteries penetrate all parts of the body, so do sympathetic fibres. The general effect of the sympathetic nervous system is to prepare the body for action in stressful situations.
The other division, the parasympathetic, or craniosacral, system, functions as the principal nerve supply to certain structures in the head. The parasympathetic system stimulates activities of the digestive organs and glands and slows the heartbeat and the respiratory rates. It tends to calm the body after a stress-producing experience, and it promotes activities that maintain life-support system.
The term “regeneration” generally stand for the ability of an organism to replace lost tissue. For example after surgical removal of a hepatic lobe, functioning new liver tissue is produced. In contrast, the nervous system usually does not form new nerve cells (neurons) after injury and therefore does not replace lost tissue. Recovery of lost function in the nervous system, when it occurs, is mediated by limited regenerative process. After section of a bundle of nerve fibres, axons may re-grow from surviving neuronal cell bodies, and eventually appropriate connections with other neurons are reformed. Successful regeneration is generally encountered if a nerve bundle is severed in the PNS. This is in sharp contrast with the course of events after lesions within the CNS. On damage to the brain or spinal cord of warm-blooded vertebrates, recovery of function is extremely limited.
Nerve growth factor (NGF) is a neurotrophic protein i.e. a special protein that controls the maintenance, size, extension of processes and transmitter synthesis in selected neurons of the PNS and CNS. Since the discovery of NGF, some 40 years ago, it provided the first evidence that nerve cells depend on specific extrinsic factors for their survival and function. Deficits of endogenous NGF or other neurothrophic factors may underlie or aggravate certain human neurodegenerative disorders, as well as apparent inability of injured adult CNS neurons to regenerate. New techniques made possible the discovery of other NGF-related trophic factors, these factors termed neutrophins (NTFs) include brain-derived neutrotrophic factor (BDNF), neurotrophins-3 (NT-3), neurotrophin 4/5 (NT-4/5), and neurotrophin-6 (NT-6). Also other neurotrophic factors unrelated to NTFs were discovered: such as insulin-like growth factors 1 and 2 (IGF-1, IGF-2), fibroblast growth factors (FGFs) and another neutrotrophic factor family, which includes ciliary neurotrophic factor (CNTF), leukaemia-inhibiting factor (LIF), interleukin-6 (IL-6), oncostatin M and Cardiotropin-1 (CT-1) (Reviewed in Elsvier's Encyclopedya of neuroscience eds Adelman at al. 1999).
LIF, CNTF, CT-1, OSM, IL-6 and IL-11 are cytokines that use gp 130 in their respective receptor complexes as a signal-transducing component (Taga et al. 1989, Kishimoto et al. 1994, Stahl et al. 1994 and Taga et al. 1997). LIF, CNTF, CT-1 and OSM support survival of several types of neurons in vitro (Ernsberger et al. 1989, Martinou et al. 1992, Taga 1996 and Horton et al. 1998). They induce cholinergic properties in cultured autonomic neurons (Yamamori et al. 1989, Patterson 1994). CNTF induces differentiation of autonomic neurons (Ernsberger et al. 1989).
It has been suggested that IL-6 acts as a nerve survival factor. Hama et al. (1989) reported that IL-6 can act as a neurotrophic agent, independent of the action of NGF, supporting neuronal survival of cultured postnatal rat septal cholinergic neurons. It was shown that IL-6, in contrast to NGF, does not affect differentiation of cultured embryonic rat septal cholinergic neurons. Horton et al. (1998) showed evidences that differentiation of sensory neurons from progenitors cells of neural crest origin is promoted by LIF and that only later in development, IL-6 promotes the survival of cultured neurons. Kushima et al. (1992) reported that IL-6 supports the survival of septal cholinergic neurons obtained from 10-day-old rats. Il-6, however in contrast to NGF, did not induce the differentiation of embryonic rat septal cholinergic neurons.
A review of the effects of IL-6 on cells of the central and peripheral nervous system indicates that the cytokine may have protective effects on neuronal cells as well as participate in inflammatory neuro-degenerative processes (Gadient and Otten, 1996, Mendel et al, 1998). On glial cells, CNTF and LIF are much more active than IL-6 to stimulate astrocyte differentiation and there is no effect on myelin protein producing cells (Kahn and De Vellis, 1994). IL-6 was found to prevent glutamate-induced cell death in hippocampal (Yamada et al. 1994) as well as in striatal (Toulmond et al. 1992) neurons. The IL-6 mechanism of neuroprotection against toxicity elicited by N-methyl-D-aspartate (NMDA), the selective agonist for NMDA subtype of glutamate receptors, is still unknown. In fact IL-6 was found to enhance the NMDA-mediated intracellular calcium elevation. In transgenic mice expressing higher levels of both IL-6 and soluble IL-6R (sIL6-R), an accelerated nerve regeneration was observed following injury of the hypoglossal nerve as shown by retrograde labeling of the hypoglossal nuclei in the brain (Hirota et al, 1996). In that work, the addition of IL-6 and sIL-6R to cultures of dorsal root ganglia (DRG) cells showed increased neurite extension in neurons, but no effect on myelinating cells or nerve generation from stem cells was reported.
Marz et al. (1998) show that in the PC-12 cell line (Greene et al. 1976), which is a tumor-derived line from a transplantable rat pheochromocytoma (vascular tumor of chromaffin tissue of the adrenal medulla or sympathetic paraganglia), only the combination of IL-6R and IL-6 but not IL-6 alone induces neuron specific differentiation. This result is not in line with the fact that these cells do express the IL-6 receptor.
As mentioned above, CNTF and LIF are cytokines acting through a common receptor system which comprises the LIF receptor (LIFR) and the gp130 chain, the latter being also part of the Interleukin-6 (IL-6) receptor complex (Ip et al, 1992). CNTF and LIF are, therefore, part of the IL-6 family of cytokines. In the case of CNTF and LIF, signal transduction operates through dimerization of LIFR with gp130, whereas in the case of IL-6 the signal is generated by the dimerization of two gp130 chains (Murakami et al, 1993). In order to bind gp130, IL-6 complexes with an IL-6 Receptor chain, which exists on certain cells as a gp80 transmembrane protein, but whose soluble form can also function as an IL-6R agonist when provided from outside the cell (Taga et al, 1989, Novick et al, 1992). By fusing the entire coding regions of the cDNAs encoding the soluble IL-6 receptor (sIL-6R) and IL-6, a recombinant IL6R/IL6 chimera can be produced in CHO cells (Chebath et al, 1997, WO99/02552). This IL6R/IL6 chimera has enhanced IL-6-type biological activities and it binds with a much higher efficiency to the gp130 chain in vitro than does the mixture of IL-6 with sIL-6R (Kollet et al, 1999).
The involvement of IL6R/IL6 in the differentiation of myelinating cells was first observed and studied in a melanoma cell line F10.9, where IL6R/IL6 chimera treatment caused transdifferentiation into myelinating Schwann cells (Chebath et al. 1997). This process involved growth arrest, loss of melanin synthesis and increase in glial cell markers. IL6R/IL6 chimera has been recently shown to induce myelinating genes e.g. MBP and P0, in embryonic Schwann cells (SC) precursors and in various tumor cells of neural crest origin (Chebath et al. 1997, Haggiag, et al. 1999, Haggiag et al, J. Neurosci. Res. 2001).
It has been observed that embryonic rat DRG explants cultured in the presence of IL6R/IL6 chimera form foci of cells with characteristic multipolar (stellar) dendritic extensions, as seen in Schwann cells or oligodendrocytes. These Schwann cell-like cells were isolated and their growth was found to be dependent on IL6R/IL6. Subclones of these cells were prepared and named CH cells (Haggiag et al. 1999). The CH cells are populations of Schwann cell precursors, which can differentiate into two directions: either myelinating Schwann cells or smooth muscle cells. When maintained without IL6R/IL6, cells of CH cell clone 1D11 have a flat morphology and slow growth, and stain for Smooth Muscle Actin (SMA). In contrast, when the CH cells are treated by IL6R/IL6, most cells differentiate into the Schwann cell phenotype and SMA is not expressed anymore. Upon treatment of CH cells with IL6R/IL6, the expression of myelin gene P0 and MBP are induced, and Pax-3 expression is repressed, indicating differentiation toward myelinating SC. When CH cells or SC are co-cultured with mouse neurons purified from DRGs, IL6R/IL6 promotes the binding of these cells along the axons, and the synthesis of P0 myelin protein (Haggiag et al, J. Neurosci. Res. 2001).
Application PCT/IL00/00363 relates to the use of IL6R/IL6 chimera for the manufacture of a medicament to generate myelinating cells or to stimulate, enhance or acceleration of myelinating cells and to induce, enhance, prolong or accelerate neuroprotection and to reduce or decelerate neuronal death.
Cells isolated from embryonic E10.5 rat neural tubes have been shown to undergo multiple rounds of self-renewing divisions in culture, and differentiate into neurons, Schwann cells, and smooth muscle-like myofibroblasts (Shah et al 1996). These cells have been termed neural crest stem cells (NCSCs). Cells with similar properties were isolated from uncultured E14.5 fetal rat sciatic nerve, using specific cell surface antibodies (Morrison et all 999). Such sciatic nerve-derived NCSCs (sNCSCs) are multipotent and self renew both in vivo and in vitro. They respond appropriately to instructive differentiation signals such as bone morphogenetic protein-2 (BMP2) and glial growth factor-2 (GGF2)/neuregulin-1 (Nrg1) (Shagh et al. 1994, 1996, 1997 and Morrison et al. 1999). These data suggest that multipotent, self renewing stem cells migrate from the neural crest of the neural tubes to peripheral tissues and continue to self renew in the peripheral tissues late into gestation.
The potential of post migratory sNCSCs isolated from rat fetal sciatic nerve was studied by direct transplantation in vivo into chick embryos (White et al. 2001). Bone morphogenetic protein-2 (BMP2) induces differentiation of sNCSCs into nerve cells and it is suggested that the choice of differentiation of these cells to either sympathetic or parasympathetic fates may be determined by the local concentration of this factor.
Autonomic neurons, Schwann (glial) cells and smooth muscle develop from the neural crest cells (Stemple et al. 1992). Three growth factors are known to promote differentiation along each of these three lineages, respectively: bone morphogenetic protein 2 (BMP2), glial growth factor 2 [GGF2, a neuregulin], and transforming growth factor β1 (TGF-β1) (Shah et al 1994 and 1996). Clonal analysis and serial observation of identified cells has suggested that each of these factors acts instructively rather than selectively on NCSCs [although some of the factors may do both (Dong et al. 1995)] i.e. GGF2, BMP2, and TGF-β1 individually direct the differentiation rather than the survival or proliferation of the majority of individual identified NCSCs plated at clonal density. The neural crest thus represents one of the few systems in which instructive lineage determination signals for multipotential stem cells have been identified (Morrison et al 1997).
Experiments have been performed in which NCSCs are exposed to different combinations of instructive signals. Depending upon the specific combination and concentration of signals tested, the experiments showed that either (i) the influence of one signal could dominate over others or (ii) the signals could exert equivalent influences, producing a mixture of lineage-committed progeny. These data therefore suggest that stem cell fate is not solely determined by what factors are present in the environment but is also influenced by cell-intrinsic differences in the relative sensitivity and timing of responses to different environmental signals (Shah et al. 1997).
Neural stem cells exist not only in the developing mammalian (embryonic) nervous system but also in the adult nervous system of all mammalian organisms, including humans. Dividing cells in the adult mouse subventricular zone (SVZ) continuously self-renew and give rise to progenies that migrate to the olfactory cortex, where they differentiate into astrocytes, oligodendrocytes and neurons (Altman et al. 1966 and Lois et al. 1994).
The function of these stem cells in the adult nervous system is uncertain. One possibility is that they are vestiges of evolution from more primitive organisms or an alternative view is that the adult mammalian nervous system retains a limited capacity for self-renewal that is important for its normal functions, like learning and memory. It is possible that the local generation of new neurons in structures could participate in the formation or integration of new memories. The ability of adult neurogenesis to be regulated by changes in the environment further supports a role in normal behavior. The implications would be that the brain controls behavior and behavior can change the structure of the brain (Gage et al. 2000).
Patent application WO0066188 discloses xenotransplant of choroid plexus cells from a neonatal mammal to provide a steady state supply of trophic factors for administration to a central nervous system in need of treatment for a neurological disease. The choroid plexus is well-innervated vascular tissue (more correctly an organ) covered with a basement membrane comprising the usual variants of collagen, one or more types of laminin proteoglycans and other extracellular matrix molecules, which is in turn covered by a unicellular epithelium-like layer and occurring in several consistent sites within the cerebral 230 ventricles. It appears to act as the source of most of the cerebrospinal fluid.
WO0066188 discloses a pharmaceutical composition, comprising an implant for implantation into the brain of a recipient mammal suffering from neurological disease, wherein the implant comprises living cells, derived from epithelial cells of the choroids plexus of another mammal, and the living cells are capable of expressing at least one product having a beneficial effect on the neurological disease into the brain of the recipient mammal. This patent specification describes the supply of neurothrophic factors from the implant to prevent deficits of endogenous NGF or other neurothrophic factors, which may underlie or aggravate certain human neurodegenerative disorders, as well as apparent inability of injured adult CNS neurons to regenerate.
Thus there exists a need to provide a therapeutic composition allowing nerve replacement in patients suffering from a neurological disease.